Transformation has been of key importance in the history of bacterial genetics in general and streptococcal genetics in particular and it is currently still the most important technique for genetic analysis of Streptococcus pneumoniae. Transformation is the major means of spread of antibiotic resistance in this species and the factors governing transformation in natural environments will be a key factor in the success of future vaccines and interventions. The goal of this grant is to address a simple question: What is the proportion of genomic information typically exchanged during transformation in the pneumococcus? Estimates based on experiments from the 60's and 70's are that this proportion is as high as 10% of the genome but those studies were based on a small number of marker genes and the results were not representative of the whole. The readout for Igenomic exchange in our experimental approach will be the proportion of strain-specific genes exchanged las measured by microarray. In Aim 1, some of the parameters that determine this proportion during in I vitro transformation will be explored, including the length of donor DNA fragment and the concentration of donor DNA. In Aim 2, gene exchange from in vivo transformation will be followed retrospectively by looking at its extent following clonal divergence. For this purpose, the clonal complex (CC14) that includes serogroup 6 isolates in five world-wide multi drug-resistant lineages from 23 countries, will be studied. CC14 is associated with invasiveness in humans and mouse virulence vary across the lineage. Keeping in mind that virulence is multi factorial, this allows the detection of virulence-associated genes in the strain-specific gene sets. In Aim 3, in vivo transformation that has occurred during nasopharyngeal carriage in a mouse model will be examined. Isolates with recombinant genotypes will be detected by FACS sorting using markers to detect exchange in genes for two of the major virulence factors, capsule and pspA (pneumococcal surface protein A). By comparing the virulence of isolates within CC14 with their genes detected using a special microarray of strain-specific genes, we hope to also identify new virulence genes of pneumococci.